Effect of dietary zinc deficiency on hematological and biochemical parameters and concentrations of zinc, copper, and iron in growing rats

Toxicology 167 (2001) 163– 170 www.elsevier.com/locate/toxicol

Effect of dietary zinc deficiency on hematological and biochemical parameters and concentrations of zinc, copper, and iron in growing rats Hassan A. El Hendy a,*, Mokhtar I. Yousef b, Nasser I. Abo El-Naga a a

b

Department of Home Economics, Faculty of Agriculture, El-Shatby, Alexandria Uni6ersity, Alexandria, Egypt Department of En6ironmental Studies, Institute of Graduate Studies and Research, Alexandria Uni6ersity, Alexandria, Egypt Received 26 September 2000; accepted 15 February 2001

Abstract Zinc has a wide spectrum of biological activities and its deficiency has been related to various dysfunctions and alterations of normal cell metabolism. The effects of adequate Zn level (38 mg/kg diet, control) and two low levels that create Zn deficiencies (19 mg/kg diet, 1/2 of control and 3.8 mg/kg diet, 1/10 of control) were investigated in growing male and female rats for 10 weeks. This allowed for evaluation of the effects these Zn levels may have on body weight gain, specific organ weights, blood parameters, and serum concentrations of Zn, Cu and Fe. Rats fed Zn-deficient diets gained less (P B0.05) than the control groups. There was increase (P B 0.05) in liver and spleen weights, and a decrease (PB 0.05) in testes weight. However, brain, kidney, heart, and lung weights were not affected (PB 0.05). Hematological parameters that were decreased (PB0.05) by Zn deficiency included hemoglobin (Hb), total erythrocyte count (TEC) and packed cell volume (PCV) with the magnitude being dose-dependent. Serum concentrations of total protein, globulin, glucose, and high density lipoprotein (HDL) also decreased (P B 0.05) in a dose-dependent manner. Zn deficiency increased (PB 0.05) total leukocyte count (TLC) and concentrations of serum albumin, total lipids, cholesterol, triglycerides and low density lipoprotein (LDL) in a dose-dependent manner. Serum concentrations of urea and creatinine were, however, not affected (P B0.05) by zinc deficiency. Zn-deficient rats had lower serum concentrations of Zn, Cu and Fe. These results showed that Zn deficiency has negative effects on growth rate, specific organ weights, hematological parameters, and serum levels of Zn, Cu and Fe, especially in rats fed the lowest Zn level. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Zinc deficiency; Growing rats; Hematological parameters; Mineral concentrations

1. Introduction Zinc deficiency in humans is common and is more prevalent in areas where the population * Corresponding author.

subsists on cereal proteins. Marginal and moderate growth impairment in children as a consequence of inadequate Zn intake has been found in many developed and developing countries (Prasad, 1996). Low consumption of foods rich in bioavailable Fe and Zn such as meat, particularly

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red meat, and high consumption of foods rich in inhibitors of Fe and Zn absorption, such as phytate, certain dietary fibers and Ca, cause Fe and Zn deficiencies. Neuropsychologic impairment is one of several potential outcomes of these deficiencies (Sandstead, 2000). Zinc deficiency can be due to inadequate dietary intake, decreased absorption, increased requirements, decreased utilization, increased loss, or genetic disease (Bettger and O’Dell, 1993; Vallee and Falchuk, 1993; Sandstead 1995). Deficiency of Zn can be observed in many disease states such as liver disease, sickle cell anemia, renal disease and gastriontestinal disorders (Cho, 1991; Vallee and Falchuk, 1993; Okada et al., 1995). The clinical manifestations of Zn deficiency include growth retardation, hypogonadism in males, neurosensory disorders, cell-mediated immunological dysfunction, and skin changes. Zinc is essential for enzymes involved in DNA synthesis, mitosis, cell division and protein synthesis. Also, it is a component of many transcription factors and proteins that control the cell cycle. Several hundreds of Zn-containing nucleoproteins are probably involved in gene expression of various proteins (Wu and Wu, 1987; Prasad, 1996; Shields et al., 1996). Deficiency of Co, Cu, Fe, I, Mn, Se, or Zn can cause a reduction in production of cattle, e.g. growth, reproduction, or lactation (Graham, 1991). Zinc is a very important element in the reproductive cycle of many species. In humans, it is necessary for the formation and maturation of spermatozoa, for ovulation, and for fertilization (Favier, 1992). Zinc supplementation has already proven beneficial in male sterility and in reducing complications during pregnancy (Favier, 1992). Zinc is essential in many biochemical processes such as the control of both cell proliferation and degeneration (Bettger and O’Dell, 1993). Zinc deficiency affects cell cycle progression (Prasad et al., 1996), although an increase in cell death without alterations in cell cycle has also been reported in embryonal Zn deficiency (Rogers et al., 1995) Because there are no enough studies carried out on the effects of high zinc deficiency on physiological performance of growing animals, therefore, the objective of this study was to

investigate the effects of two low levels that create Zn deficiencies on growth rate, protein synthesis, hematological parameters, and serum concentrations of Zn, Cu, and Fe in growing rats.

2. Materials and methods

2.1. Animals and treatments Forty-two weaned male and female albino rats (1-month old and weighing 42.79 0.54 g) were used. The ethics committee of Alexandria University has approved the design of the experiment and the experimental protocol followed the NIH guidelines. Animals were caged in groups and given food and water ad libitum throughout the 10-week experimental period. After a period of acclimation, animals were divided into three equal groups of 14 animals each (seven males and seven females). The first group (control) was fed the requirement of zinc, 38 mg Zn/kg basis diet (Ruz et al., 1999), the second group (1/2 of control) was fed a Zn-deficient diet (19 mg Zn/kg basis diet) and the thrid group (1/10 of control) was fed a very low Zn diet (3.8 mg Zn/kg basis diet). Rats received a purified diet (Table 1) with dried skim milk being the protein source. Body weight was recorded at the beginning of the experiment and weekly during the experimental period. At the end of the experimental period, rats were sacrificed by cervical decapitation and the brain, heart, lungs, spleen, kidney, testes, and liver were weighed. The organ to body weight ratio was calculated. Table 1 Composition of the experimental diet Ingredient

Amount (g/kg)

Dried skim milk Corn oil Corn starch Sucrose Cellulose Vitamin mixture Mineral mixture

375 90 300 225 5 1 4

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2.2. Hematological and biochemical parameters

2.3. Zn, Cu and Fe analysis

At the end of the experiment, blood samples were collected from the sacrificed animals for separation of plasma and serum. Plasma and serum from blood tubes with or without heparin, respectively, were obtained by centrifugation at 860×g for 20 min, stored at −20°C. Non-coagulated blood was tested, shortly after collection, for hemoglobin (Hb), total erythrocyte count (TEC), packed cell volume (PCV) and total leukocyte counts (TLC). Blood Hb concentrations were determined by the cyanomethemoglobin procedure (Wintrobe, 1965) using commercial kit, Modern Laboratory Chemicals, Diamond Diagnostics, Alexandria, Egypt. Erythrocytes were counted on AO Bright line hemocytometer using a light microscope at 40×10 magnification. Blood samples were diluted to 200 times by physiological saline (0.9% NaCl solution) before counting. Micro Wintrobe hematocrit tubes and a hematocrit centrifuge (at 4000 rpm for 5 min) were used to determine volume PCV or hematocrit value. Leukocytes were counted on AO Bright line hemocytometer using a light microscope at 10× 10 magnification after diluting the samples to 20 times with 1% acetic acid solution containing trace of Leishman’s stain (Hepler, 1966). Stored serum samples were analyzed for total protein (TP) by the Biuret method (Henry et al., 1974). Albumin concentrations were determined by the method of Doumas et al. (1977). Globulin concentrations were determined by difference (total protein −albumin). Plasma glucose content was measured (Trinder, 1969) and serum concentration of urea and creatinine were determined by the methods of Patton and Crouch (1977) and Henry et al. (1974), respectively. Serum concentration of total lipids, cholesterol, and triglycerides (TG) also were determined according to the methods of Knight et al. (1972), Watson (1960) and Fossati and Principe (1982), respectively. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) were determined according to the methods of Warnick et al. (1983) and Bergmenyer (1985), respectively.

The concentrations of Zn, Cu, and Fe in serum were determined (Parker et al., 1967) by using an atomic absorption spectrophotometer (Perkin– Elmer 3300, 761 Main Ave., Norwalk, CT 068590012, USA).

2.4. Statistical analysis Data were analyzed by least-squares analysis. Male and female data were analyzed separately using completely randomized design (Steel and Torrie, 1980). The analyses were conducted according to the General Linear Model procedure of SAS (1995). Difference between each two dose means within each sex was compared with LSD at 0.05 significant level (Steel and Torrie, 1980).

3. Results and discussion

3.1. Effect of zinc deficiency on body weight and organ to body weight ratio Zinc is required for the normal growth and development of all animal species, including humans and its deficiency results in reducing growth rate (Prasad, 1993). As previously reported (Mengheri et al., 1995), Zn deficiency had a dramatic effect on body weight of rats. In the present study, male and female rats fed 1/2 and 1/10 of zinc adequate weighed 39, 42, 70 and 69% less than the control rats, respectively, (Table 2). The changes in relative weights of brain, heart, lung, spleen, kidney, testes and liver of male and female rats fed on Zn adequate, 1/2 and 1/10 of Zn adequate are presented in Table 2. The present data indicated an increase (PB 0.05) in relative weights of liver and spleen of both sexes fed 1/2 and 1/10 of Zn adequate. While the relative weight of testes decreased (PB 0.05) by 20 and 35% of 1/2 and 1/10 Zn of control, respectively. On the other hand, the relative weights of brain, heart, lung and kidney did not change. These findings are in agreement with those of others (Hafiez et al., 1990; Ai et al., 1997; Kraus et al., 1997; Stallard and Reeves, 1997) in Zn-deficient

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Table 2 Mean of body and relative organ weights of male and female rats fed on zinc deficiency (1/2 and 1/10 of Zn adequate)a Parameter

Control

1/2 Zn

1/10 Zn

S.E.M.

Male Weight gain g/10 week Brain (g/100 g b.w.) Heart (g/100 g b.w.) Lung (g/100 g b.w.) Spleen (g/100 g b.w.) Kidney (g/100 g b.w.) Testes (g/100 g b.w.) Liver (g/100 g b.w.)

131.2b 1.75b 0.5 1.01 0.54b 1.0 0.97b 3.61b

98.8c 1.88b 0.51 1.06 0.62c 0.95 0.77c 3.86b,c

43.6d 1.57c 0.55 1.13 0.73d 0.99 0.63c 4.25c

2.35 0.04 0.04 1.04 0.02 0.07 0.06 0.12

Female Weight gain g/10 week Brain (g/100 g b.w.) Heart (g/100 g b.w.) Lung (g/100 g b.w.) Spleen (g/100 g b.w.) Kidney (g/100 g b.w.) Liver (g/100 g b.w.)

137.1b 1.64 0.5 1.11 0.49b 1.0 3.66b

81.0c 1.71 0.48 1.04 0.57b,c 1.0 3.92b,c

46.6d 1.73 0.47 1.19 0.68c 1.1 4.17c

2.73 0.07 0.11 0.07 0.06 0.07 0.09

a

Values are the means of seven rats. within rows, between control and treated animals, means with different letters differ significantly (PB0.05).

b,c,d

human and rats. MacDonald et al. (1998) suggested that growth failure in Zn-deficient animals is associated with decreased DNA synthesis. Levin et al. (1992) reported that reduction of voluntary food intake is a common symptom with Zn-deficiency and reduced appetite. Zinc requirements in total diet of beef cattle, dairy cattle, swine, horses, sheep, goats, chickens, and turkeys are 30, 40, 50, 40, 20– 33, 40– 75, 40– 50, and 40–75 ppm, respectively, (NRC, 1978a,b, 1981, 1988). In a study with sheep, throughout pregnancy unsupplemented pregnant ewes gained less weight than the Zn-supplemented ones (Apgar and Fitzgerald, 1985). The weight of newly born lambs from Zn-deficient ewes also was 20% less than those from Zn-supplemented ewes. Essman (1987) reported that Zn deficiency could affect taste. Martin and White (1992) indicated that appetite of Zn-deficient rams was half of that the controls. Stallard and Reeves (1997) found that Zn-deficient rats had lower daily weight gain and testes weight than those with Zn-adequate diets. Underwood (1981) reported that growth retardation and delayed onset of puberty were common in Zn-deficient bull calves. This was due, in part,

to decreased appetite and impaired protein synthesis. Chhabra et al. (1990) reported that the increase in spleen weight of rat, after treatment with p-chloroaniline, was due to excessive deposition of damaged erythrocytes. Kraus et al. (1997) also found that Zn-deficient rats (1.1 mg Zn/kg diet) showed increased osmotic fragility of erythrocytes.

3.2. Hematological and biochemical parameters The effects of Zn deficiency on some hematological parameters in rats are presented in Table 3. These data showed a significant decline (PB 0.05) in Hb, TEC and PCV of both male and female rats that fed on 1/10 of Zn adequate. This response indicate the development of a mild to moderate anemia. Reduction in Hb content may be due to increased rate of disruption or reduction in the rate of formation of erythrocyte (Shakoori et al., 1992). Recorded low TEC in this group fed the lowest level of Zn, supports this hypothesis. The decrease in PCV was obviously due to the decreased cellular count in blood of rats that were Zn deficient. Kraus et al. (1997) reported that

H.A. El Hendy et al. / Toxicology 167 (2001) 163–170 Table 3 Means of blood hemoglobin (Hb), total erythrocyte counts (TEC), packed cell volume (PCV) and total leukocyte count (TLC) of male and female rats fed on zinc deficiency (1/2 and 1/10 of Zn adequate)a Parameters Male Hb (g/100 ml) RBC (×106/mm3) PCV (%) TLC (×103/mm3) Female Hb (g/100 ml) RBC (×106/mm3) PCV (%) TLC (×103/mm3)

Control

1/2 Zn

1/10 Zn

S.E.M.

12.3b 4.5b

11.7b 4.1c

10.2c 3.5d

0.17 0.05

35.6b 6.5b

31c 6.6b

29.6c 7.7c

0.58 0.46

12.3b 5.5b

11.3c 4.6c

9.7d 4.1c

0.19 0.17

31.4b 5.9b

27.8c 6.6c

26.2c 8.2c

0.81 0.23

a

Values are the means of seven rats. within rows, between control and treated animals, means with different letters differ significantly (PB0.05). b,c,d

dietary Zn deficiency in rats increased osmotic fragility of their erythrocytes and the oxidative damage was responsible for the impaired erythrocyte stability. Shi et al. (1999) found that Zn deficiency reduced numbers of spleen cells. Shakoori et al. (1990) suggested that the decrease

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in red blood cells (RBC) of rabbits was either indicative of excessive damage to erythrocytes or inhibition of erythrocyte formation. Shakoori et al. (1994) suggested that the reduction in RBC and Hb content of rabbits could be probably due to the blockage of protein synthesis and histogenesis. Since Zn is essential for integrity of the immune system, deficiency results in reduced immunocompetence and decreased resistance to infections (Prasad, 1993). Results illustrated in Table 3 showed a significant increase (PB0.05) in TLC of male and female rats fed the lowest level of Zn (1/10 of control). A deficiency of Zn affects proliferation and maturity of lymphocytes adversely (Prasad, 1996). Lepage et al. (1999) reported that compromised immune function is common with Zn deficiency. Also, Shi et al. (1999) found that zinc deficiency caused an impairment of immune responses. This increase in leukocyte counts may indicate an activation of the animal’s defense mechanism and immune system. Zinc is essential for synthesis of coenzymes that mediate biogenic-amine synthesis and metabolism (Sandstead et al., 2000). Male and female rats fed the lowest Zn level (1/10 of control) showed a reduction (PB 0.05) in serum total protein and globulin, and the magnitude of reduction was 27 and 54 of males, 24 and 52 of female compared

Table 4 Means of serum total protein (TP), albumin (A), globulin (G), urea and creatinine concentrations of male and female rats fed on zinc deficiency (1/2 and 1/10 of Zn adequate)a Parameters

Control

1/2 Zn

1/10 Zn

S.E.M.

Male TP (g/100 ml) A (g/100 ml) G (g/100 ml) Urea (mg/100 ml) Creatinine (mg/100 ml)

8.17b 2.96b 5.2b 14.58 3.48

7.43b 2.92b 4.51b 14.48 3.59

6.0c 3.5c 2.41c 15.91 4.03

0.19 0.15 0.26 0.46 0.11

Female TP (g/100 ml) A (g/100 ml) G (g/100 ml) Urea (mg/100 ml) Creatinine (mg/100 ml)

8.19b 3.23b 4.96b 14.79 3.38

7.82b 3.42b 4.4b 15.41 3.13

6.23c 3.86c 2.38c 15.55 3.82

0.20 0.26 0.32 0.57 0.10

a

Values are the means of seven rats. within rows, between control and treated animals, means with different letters differ significantly (PB0.05).

b,c

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Table 5 Means of plasma glucose, serum total lipids (TL), cholesterol, triglycerides (TG), high density of lipoproteins (HDL) and low density of lipoproteins (LDL) of male and female rats fed on zinc deficiency (1/2 and 1/10 adequate)a Parameters

Control

1/2 Zn

1/10 Zn

S.E.M.

Male Glucose (gm/100 ml) Total lipids (mg/100 ml) Cholesterol (mg/100 ml) TG (mg/100 ml) HDL (mg/100 ml) LDL (mg/100 ml)

94.4b 439b 117.1b 142.3b 45.7b 49.0b

47.6c 575c 142.8c 180.2b 41.0b 65.8b

37.6c 667d 159.5d 228.9c 35.4c 76.8c

4.56 27.23 7.47 11.03 1.41 5.76

Female Glucose (gm/100 ml) Total lipids (mg/100 ml) Cholesterol (mg/100 ml) TG (mg/100 ml) HDL (mg/100 ml) LDL (mg/100 ml)

116.4b 455b 119.7b 94.6b 60.2b 44.7b

83.3c 539c 140.2c 108.0b 52.9b 63.1b

51.0d 699d 153.0d 161.3c 35.3c 79.8c

4.19 26.0 4.40 7.33 3.86 5.66

a

Values are the means of seven rats. within rows, between control and treated animals, means with different letters differ significantly (PB0.05).

b,c,d

with control, respectively, (Table 4). Serum albumin, however, was elevated (P B0.05) in both male and female rats fed the lowest Zn level. Serum urea and creatinine concentrations were not affected by reducing Zn level in the diet. The findings of Prasad (1996) showed that Zn deficiency influences DNA synthesis, cell division and protein synthesis. Also, Eltohamy and Younis (1991) reported significant reductions in the levels of protein of both testes and epididymis of Zndeficient rabbits. In addition, Oteiza et al. (1996) demonstrated that zinc deficiency could be associated with high rates of oxidative damage to proteins and DNA in male rats. Table 5 shows the changes in plasma glucose and serum total lipids, cholesterol, TG, HDL and LDL of growing male and female rats fed Zn-deficient diets (1/2 or 1/10 of control). Plasma glucose and serum HDL were decreased (P B 0.05) in Zn-deficient males and females in a dose-dependent manner. Serum total lipids, cholesterol, TG, and LDL, however, were increased (P B 0.05) in Zn-deficient rats in a dose-dependent manner (Table 5). Lipid composition and essential fatty acids concentration are altered in the brain of zinc-deficient animals (Essman, 1987). Eltohamy and Younis (1991) found that there was signifi-

cant increase in the cholesterol and glycogen contents of rabbit testes. Chausmer (1998) reported that Zn play a clear role in the synthesis, storage and secretion of insulin in human as well as conformational integrity of insulin in the hexameric form.

3.3. Concentrations of Zn, Cu, and Fe status in serum Serum concentrations of Zn, Cu, and Fe were significantly decreased in both male and female rats that were fed the Zn-deficient diets (Table 6). The percentage reductions in Zn ranged from 17 to 78%, and in Cu from 37 to 51%, and in Fe from 30 to 78% compared with the control group. These results are in agreement with those reduction reported by Martin and White (1992). Apgar and Fitzgerald (1985) reported that ewes fed Zndeficient diets maintained lower plasma Zn levels than Zn-supplemented ewes throughout gestation. Zinc deficiency in women, infants, and children also decreased serum Zn levels (Prasad, 1996). Also, reductions were observed in plasma and urinary Zn of Zn-deficient humans (Ruz et al., 1991). In addition, Eltohamy and Younis (1991) reported significant reductions in the levels of Zn

H.A. El Hendy et al. / Toxicology 167 (2001) 163–170 Table 6 Zinc, copper and iron status in serum of male and female rats fed on zinc deficiency (1/2 and 1/10 of Zn adequate)a Parameters

Control

1/2 Zn

1/10 Zn

S.E.M.

Male Zinc (mg/l) Copper (mg/l) Iron (mg/l)

9.4b 0.40b 4.02b

6.2c 0.25c 2.27c

2.1d 0.22c 0.88d

0.18 0.02 0.06

Female Zinc mg/l) Copper (mg/l) Iron (mg/l)

8.7b 0.51b 4.15b

7.2c 0.31c 2.89c

2.3d 0.25c 1.10d

0.17 0.03 0.17

a

Values are the means of seven rats. within rows, between control and treated animals, means with superscript letters differ significantly (PB0.05). b,c,d

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